Zwitterionic Hydrophilic Interaction Chromatography Research Articles

Investigation of several chromatographic approaches for untargeted profiling of central carbon metabolism

Monitoring the central carbon metabolism (CCM) network using liquid chromatography/mass spectrometry (LC-MS) analysis is hampered by the diverse chemical nature of its analytes, which are extremely difficult to analyze using single chromatographic conditions. Furthermore, CCM-related compounds present non-specific adsorption on metal surfaces, causing detrimental chromatographic effects and sensitivity loss. In this study, polar reversed-phase, mixed-mode (MMC), and zwitterionic hydrophilic interaction chromatography (HILIC) featuring low-adsorption hardware were investigated towards untargeted analysis of biological samples with a focus on energy metabolism-related analytes. Best results were achieved with sulfoalkylbetaine HILIC with different supports, where polymeric option featured the highest coverage and inert hybrid silica facilitated best throughput and kinetic performance at a cost of less selectivity for small carboxylic acids. MMC demonstrated excellent performance for strongly anionic analytes such as multiresidue phosphates. The obtained experimental data also suggested that an additional hydrophilic modulation might be necessary to facilitate better resolution of carboxylic acids in zHILIC mode, as found during the application of the developed method to study the effect of two different mutations on the energy metabolism of S. aureus.

Comprehensive evaluation of zwitterionic hydrophilic liquid chromatography stationary phases for oligonucleotide characterization

Hydrophilic interaction chromatography (HILIC) has been proposed as a valuable alternative to ion-pairing reversed-phase chromatography (IP-RPLC) for oligonucleotide (ON) analysis. In this context, the potential of seven zwitterionic HILIC columns has been evaluated against amide- and poly-hydroxy fructan-functionalized HILIC columns and a C18 column operated under IP-RPLC mode.Based on the retention characteristics of key small molecule pairs, each zwitterionic HILIC column showed a unique radar-shaped profile, suggesting different selectivities for distinct structural differences. Unmodified DNA and RNA samples were then evaluated, and the columns classified based on their retentivity. Two zwitterionic columns were particularly promising in terms of overall resolution, especially for the largest ONs (> 40-mer). Finally, separations between a chemically modified drug-like ON and its closely related impurities were performed. Although the ZIC-cHILIC column showed similar selectivity values as compared to the reference IP-RPLC technique, all columns demonstrated a general decrease in selectivity due to the minor structural differences present in the highly complex samples.This work highlights the utility of zwitterionic HILIC mode for ON analysis and it reveals the importance of understanding columns characteristics – in terms of retention and selectivity – when selecting a stationary phase for specific ON applications.

Characterization of a highly stable zwitterionic hydrophilic interaction chromatography stationary phase based on hybrid organic-inorganic particles.

We have characterized a sulfobetaine stationary phase based on 1.7 μm ethylene‐bridged hybrid organic–inorganic particles, which is intended for use in hydrophilic interaction chromatography. The efficiency of a column packed with this material was determined as a function of flow rate, demonstrating a minimum reduced plate height of 2.4. The batch‐to‐batch reproducibility was assessed using the separation of a mixture of acids, bases, and neutrals. We compared the retention and selectivity of the hybrid sulfobetaine stationary phase to that of several benchmark materials. The hybrid sulfobetaine material gave strong retention for polar neutrals and high selectivity for methyl groups, hydroxy groups, and configurational isomers. Large differences in cation and anion retention were observed among the columns. We characterized the acid and base stability of the hybrid sulfobetaine stationary phase, using accelerated tests at pH 1.3 and 11.0, both at 70°C. The results support a recommended pH range of 2–10. We also investigated the performance of columns packed with this material for metal‐sensitive analytes, comparing conventional stainless steel column hardware to hardware that incorporates hybrid surface technology to mitigate interactions with metal surfaces. Compared to the conventional columns, the hybrid surface technology columns showed a greatly improved peak shape.

ZIC-cHILIC-Based StageTip for Simultaneous Glycopeptide Enrichment and Fractionation toward Large-Scale N-Sialoglycoproteomics.

Alterations of protein glycosylation are closely related with pathophysiological regulation. Due to the structural macro- and microheterogeneity, low stoichiometry, and low ionization efficiency of glycopeptides, high-performance tools to enrich glycopeptides, especially the negatively charged and labile sialoglycopeptides, are essential to enhance the identification of the underexplored glycoproteome. Here, we present the first implementation of zwitterionic hydrophilic interaction chromatography with the exposed choline group (ZIC-cHILIC) in StageTip for simultaneous enrichment and fractionation of intact glycopeptides. In a model study using lung cancer cells, early elution by a high percentage of acetonitrile prominently prefilters nonglycopeptides, facilitating high enrichment specificity for glycopeptides (92-96%) and sialoglycopeptides (77-89%) in the subsequent hydrophilic fractions. The stepwise elution shows a high glycopeptide fractionation efficiency by a <10% overlap of glycopeptides between adjacent fractions. Most importantly, the ZIC-cHILIC stepwise strategy demonstrated good reproducibility (>80% in triplicate analysis) as well as superior coverage of 4.6- to 12.0-fold and 2.1- to 35.6-fold more glycopeptides and sialoglycopeptides compared to conventional TiO2 and ZIC-HILIC, respectively. To the best of our knowledge, the result with 2742 sialoglycopeptides among 7367 unique glycopeptides and 166 glycans from 2434 N-glycosites of 1118 glycoproteins (Byonic score > 100) provides one of the deepest glycoproteomic profiles in single-cell type. Without the immunoprecipitation step, the large-scale glycoproteomic atlas also reveals site-specific glycosylation of many druggable receptor proteins, such as EGFR, MET, ERBB2, ERBB3, AXL, and IGF1R. The demonstrated high enrichment specificity and identification depth show that stepwise ZIC-cHILIC is an efficient method to explore the under-represented sialoglycoproteome.

Site-Specific N- and O-Glycosylation Analysis of Human Plasma Fibronectin

Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the 18O-labeling method. O-glycopeptide enrichment and O-glycosite identification were achieved by an enzyme-assisted site-specific extraction method. An RP–LC–MS/MS system functionalized with collision-induced dissociation and stepped normalized collision energy (sNCE)-HCD tandem mass was applied to analyze the glycoforms of fibronectin. A total of 6 N-glycosites and 53 O-glycosites were identified, which were occupied by 38 N-glycoforms and 16 O-glycoforms, respectively. Furthermore, 77.31% of N-glycans were sialylated, and O-glycosylation was dominated by the sialyl-T antigen. These site-specific glycosylation patterns on human fibronectin can facilitate functional analyses of fibronectin and therapeutics development.

Relative Quantification of Phosphorylated and Glycosylated Peptides from the Same Sample Using Isobaric Chemical Labelling with a Two-Step Enrichment Strategy.

Post-translational modifications (PTMs) are essential for the regulation of all cellular processes. The interplay of various PTMs on a single protein or different proteins comprises a complexity that we are far from understanding in its entirety. Reliable strategies for the enrichment and accurate quantification of PTMs are needed to study as many PTMs on proteins as possible. In this protocol we present a liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based workflow that enables the enrichment and quantification of phosphorylated and N-glycosylated peptides from the same sample. After extraction and digestion of proteins, we label the peptides with stable isotope-coded tandem mass tags (TMTs) and enrich N-glycopeptides and phosphopeptides by using zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) and titanium dioxide (TiO2) beads, respectively. Labelled and enriched N-glycopeptides and phosphopeptides are further separated by high pH (basic) reversed-phase chromatography and analyzed by LC/MS/MS. The enrichment strategies, together with quantification of two different PTM types from the same sample, allow investigation of the interplay of those two PTMs, which are important for signal transduction inside the cell (phosphorylation), as well as for messaging between cells through decoration of the cellular surface (glycosylation).

Site- and structure-specific characterization of the human urinary N-glycoproteome with site-determining and structure-diagnostic product ions.

N-glycosylation is one of the most common protein post-translational modifications; it is extremely complex with multiple glycoforms from different monosaccharide compositions, sequences, glycosidic linkages, and anomeric positions. Each glycoform functions with a particular site- and structure-specific N-glycan that can be fully characterized using state-of-the-art tandem mass spectrometry (MS/MS) and the intact N-glycopeptide database search engine GPSeeker that we recently developed. Urine has recently gained increasing attention as a non-invasive source for disease marker discovery. In this study, we report our structure-specific N-glycoproteomics study of human urine. We performed trypsin digestion, Zwitterionic Hydrophilic Interaction chromatography (ZIC-HILIC) enrichment, C18-RPLC/nano-ESI-MS/MS using HCD with stepped normalized collisional energies, and GPSeeker database search for a comprehensive site- and structure-specific N-glycoproteomics characterization of the human urinary N-glycoproteome at the intact N-glycopeptide level. For this, we used b/y product ion pairs from the GlcNAc-containing site-determining peptide backbone and structure-diagnostic product ions from the N-glycan moieties, respectively. We identified 2986 intact N-glycopeptides with comprehensive site and structure information for the peptide backbones (amino acid sequences and N-glycosites) and the N-glycan moieties (monosaccharide compositions, sequences/linkages). The 2986 intact N-glycopeptide IDs corresponded to 754 putative N-glycan linkage structures on 419 N-glycosites of 450 peptide backbones from 327 intact N-glycoproteins. Next, 146 linkage structures and 200 N-glycosites were confirmed with structure-diagnostic and GlcNAc-containing site-determining product ions, respectively. We found 106 new N-glycosites not annotated in the current UniProt database. The elution-abundance patterns of urinary intact N-glycopeptide oxonium ions (m/z 138 and 204) of the same subject were temporally stable during the day and over 6 months. These patterns are rather different among different subjects. The results implied an interesting possibility that glycopeptide oxonium ion patterns could serve as distinguishing markers between individuals and/or between physiological and pathological states.

Mass Spectrometric Method for the Unambiguous Profiling of Cellular Dynamic Glycosylation

Various biological processes at the cellular level are regulated by glycosylation which is a highly microheterogeneous post-translational modification (PTM) on proteins and lipids. The dynamic nature of glycosylation can be studied through metabolic incorporation of non-natural sugars into glycan epitopes and their detection using bio-orthogonal probes. However, this approach possesses a significant drawback due to nonspecific background reactions and ambiguity of non-natural sugar metabolism. Here, we report a probe-free strategy for their direct detection by glycoproteomics and glycomics using mass spectrometry (MS). The method dramatically simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, N-acetyl-d-galactosamine (GalNAc) and N-acetyl-d-glucosamine (GlcNAc) biosynthetic pathways. Multistage enrichment of glycoproteins by cellular fractionation, subsequent ZIC-HILIC (zwitterionic-hydrophilic interaction chromatography) based glycopeptide enrichment, and a spectral enrichment algorithm for the MS data processing enabled direct detection of non-natural monosaccharides that are incorporated at low abundance on the N/O-glycopeptides along with their natural counterparts. Our approach allowed the detection of both natural and non-natural sugar bearing glycopeptides, N- and O-glycopeptides, differentiation of non-natural monosaccharide types on the glycans and also their incorporation efficiency through quantitation. Through this, we could deduce interconversion of monosaccharides during their processing through glycan salvage pathway and subsequent incorporation into glycan chains. The study of glycosylation dynamics through this method can be conducted in high throughput, as few sample processing steps are involved, enabling understanding of glycosylation dynamics under various external stimuli and thereby could bolster the use of metabolic glycan engineering in glycosylation functional studies.

Offline preparative 2-D polar-copolymerized reversed-phase chromatography × zwitterionic hydrophilic interaction chromatography for effective purification of polar compounds from Caulis Polygoni Multiflori

Polar compounds are abundant in the water decoction of Traditional Chinese Medicines (TCMs) and possess important biological activities. However, purifying these compounds has faced great difficulties largely due to poor retention and insufficient selectivity. To solve this problem, an offline orthogonal 2-D RPLC coupled with hydrophilic interaction chromatography (HILIC) method was developed to achieve purification of polar compounds from Caulis Polygoni Multiflori. A polar-copolymerized XAqua C18 column and a zwitterionic Click XIon column exhibited satisfactory retention and separation for polar compounds. Therefore, they were adopted to construct the offline 2-D LC system. Furthermore, the method presented a high orthogonality, which was calculated to be 72%. The XAqua C18 column was used in the first dimension to fractionate the 5.1 g polar fraction with a recovery of 85% within 10 h. In the second dimension, three representative fractions were purified using the Click XIon column. Finally, three compounds with purity higher than 95% were identified for the first time from this plant. This offline 2-D RPLC/HILIC method was shown to be an effective approach to purifying polar compounds from Caulis Polygoni Multiflori.

Analysis of Underivatized Amino Acids: Zwitterionic Hydrophilic Interaction Chromatography Combined with Triple Quadrupole Tandem Mass Spectrometry.

Analysis of underivatized amino acids is challenging regarding both separation and detection of this small, polar, and largely UV-inactive compounds. Additives for reversed phase chromatography such as ion pairing reagents can hamper mass spectrometric detection. Zwitterionic hydrophilic interaction chromatography using MS compatible eluents together with tandem mass spectrometry in multiple reaction monitoring mode for selective detection is an attractive approach to overcome the abovementioned issues.

Preparation and evaluation of chromatographic properties of sulfobetaine zwitterionic hydrophilic interaction chromatography stationary phase

Preparation and evaluation of chromatographic properties of sulfobetaine zwitterionic hydrophilic interaction chromatography stationary phase

Hydrophilic interaction liquid chromatography method development and validation for the assay of HEPES zwitterionic buffer

HEPES is a zwitterionic buffer component used as a raw material in the GMP-manufacturing of advanced therapy medicinal products (ATMPs), hence requiring an adequate assay method with sufficient selectivity toward related impurities. Therefore, a hydrophilic interaction chromatography (HILIC) method was developed. Different factors were investigated towards the retention behavior of HEPES, its analogue EPPS and its starting material isethionate: pH, ion concentration and organic solvent ratio of the mobile phase, as well as column temperature. Moreover, stress testing resulted in the N-oxide degradant, identified by high resolution MS. The final method consisted of an isocratic system with an aqueous (pH 2.0 with H3PO4) acetonitrile (35:65, v/v) mobile phase on a zwitterionic HILIC (Obelisc N) column with a flow rate of 0.5mL/min and UV detection at 195nm. The assay method of HEPES was validated, obtaining adequate linearity (R2=0.999), precision (RSD of 0.5%) and accuracy (recovery of 100.08%). Finally, the applicability of the validated method was demonstrated by analysis of samples from different suppliers.

Synthesis of Zwitterionic Polymer Particles via Combined Distillation Precipitation Polymerization and Click Chemistry for Highly Efficient Enrichment of Glycopeptide.

Because of the low abundance of glycopeptide in natural biological samples, methods for efficient and selective enrichment of glycopeptides play a significant role in mass spectrometry (MS)-based glycoproteomics. In this study, a novel kind of zwitterionic hydrophilic interaction chromatography polymer particles, namely, poly(N,N-methylenebisacrylamide-co-methacrylic acid)@l-Cys (poly(MBAAm-co-MAA)@l-Cys), for the enrichment of glycopeptides was synthesized by a facile and efficient approach that combined distillation precipitation polymerization (DPP) and "thiol-ene" click reaction. In the DPP approach, residual vinyl groups explored outside the core with high density, then the functional ligand cysteine was immobilized onto the surface of core particles by highly efficient thiol-ene click reaction. Taking advantage of the unique structure of poly(MBAAm-co-MAA)@l-Cys, the resulting particles possess remarkable enrichment selectivity for glycopeptides from the tryptic digested human immunoglobulin G. The polymer particles were successfully employed for the analysis of human plasma, and 208 unique glycopeptides corresponding to 121 glycoproteins were reliably identified in triple independent nano-LC-MS/MS runs. The selectivity toward glycopeptides of these particles poly(MBAAm-co-MAA)@l-Cys is ∼2 times than that of the commercial beads. These results demonstrated that these particles had great potential for large-scale glycoproteomics research. Moreover, the strategy with the combination of DPP and thiol-ene click chemistry might be a facile method to produce functional polymer particles for bioenrichment application.

Characterization of Protein N-Glycosylation by Analysis of ZIC-HILIC-Enriched Intact Proteolytic Glycopeptides.

Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) solid-phase extraction (SPE) combined with direct-infusion nanoESI mass spectrometry (MS) and tandem MS/MS is a well-suited method for the analysis of protein N-glycosylation. A site-specific characterization of N-glycopeptides is achieved by the combination of proteolytic digestions employing unspecific proteases, glycopeptide enrichment by use of ZIC-HILIC SPE, and subsequent mass spectrometric analysis. The use of thermolysin or a mixture of trypsin and chymotrypsin leads per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. As a result of their higher hydrophilicity N-glycopeptides comprising short peptide backbones are preferably accumulated by the ZIC-HILIC-based separation procedure. By employing this approach complications associated with low ionization efficiencies of N-glycopeptides resulting from signal suppression in the presence of highly abundant nonglycosylated peptides can be largely reduced. Here, we describe a simple protocol aimed at the enrichment of N-glycopeptides derived from in-solution and in-gel digestions of SDS-PAGE-separated glycoproteins preceding mass spectrometric analysis.

Sequential enrichment of singly- and multiply-phosphorylated peptides with zwitterionic hydrophilic interaction chromatography material

An interesting and novel method for the selective and sequential enrichment of singly- and multiply-phosphorylated peptides with a zwitterionic material “Click TE-Cys” is presented. Retention mechanisms between phosphopeptides and Click TE-Cys are systematically investigated by checking the influence of acetonitrile content, pH value, and buffer concentration on the retention of phosphopeptides. Both hydrophilic interaction and electrostatic interaction are involved in retention between phosphopeptides and Click TE-Cys. Based on these results, an optimized method is established for selective enrichment of phosphopeptides using Click TE-Cys. This method not only exhibits high selectivity for phosphopeptides, but also fractionates singly- and multiply-phosphorylated peptides into two fractions. This method was evaluated using relatively complex samples, including peptide mixtures of α-casein and bovine serum albumin (BSA) at a molar ratio of 1:10 and skim milk. This efficient and optimized protocol has great potential for enriching multiply-phosphorylated peptides and could be a valuable tool for specific enrichment of phosphopeptides in phosphoproteome analysis.

Validated Stability-Indicating Method for Alendronate Sodium Employing Zwitterionic Hydrophilic Interaction Chromatography Coupled with Charged Aerosol Detection

Alendronate sodium is widely used in the treatment of osteoporosis and Paget’s disease. The HPLC method development for alendronate sodium, in particular, is challenging owing to the absence of chromophoric group and its high polarity. In the present study, a short and simple isocratic method was developed involving hydrophilic interaction liquid chromatography, coupled with a charged aerosol detector. The developed method was validated according to the ICH Q2(R1) guideline and was successfully applied for the analysis of a marketed formulation containing the drug.

Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry

Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry (HILIC–ESI–MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90% of the analyzed metabolites presented detection limits better than 0.5pmol (on column), and 70% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC–pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites.

Shotgun metabolomic profiles in maternal urine identify potential mass spectral markers of abnormal fetal biochemistry - dihydrouracil and progesterone in the metabolism of Down syndrome.

In Down syndrome (DS) in particular, the precise cellular mechanisms linking genotype to phenotype is not straightforward despite a clear mapping of the genetic cause. Metabolomic profiling might be more revealing in understanding molecular-cellular mechanisms of inborn errors of metabolism/syndromes than genomics alone and also result in new prenatal screening approaches. The urinary metabolome of 122 maternal urine from women with and without an aneuploid pregnancy (predominantly Down syndrome) were compared by both zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) and reversed-phase liquid chromatography (RPLC) coupled to hybrid ion trap time of flight mass spectral analysis. ZIC-HILIC mass spectrometry resolved 10-fold more unique molecular ions than RPLC mass spectrometry, of which molecules corresponding to ions of m/z 114.07 and m/z 314.20 showed maternal urinary level changes that significantly coincided with the presence of a DS fetus. The ion of m/z 314.20 was identified as progesterone and m/z 114.07 as dihydrouracil. A metabolomics profiling-based maternal urinary screening test modelled from this separation data would detect approximately 87 and 60.87% (using HILIC-MS and RPLC-MS, respectively) of all DS pregnancies between 9 and 23 weeks of gestation with no false positives.

N-glycoproteome Analysis of the Secretome of Human Metastatic Hepatocellular Carcinoma Cell Lines Combining Hydrazide Chemistry, HILIC Enrichment and Mass Spectrometry

Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC) cell lines with low (MHCC97L) or high (HCCLM3) metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC), with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.

Synthesis of zwitterionic polymer brushes hybrid silica nanoparticles via controlled polymerization for highly efficient enrichment of glycopeptides

Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) materials have been increasingly attractive in glycopeptide enrichment. However, the traditional ZIC-HILIC materials are modified with monolayer zwitterionic molecules on the surface, therefore, the hydrophilicity, detection sensitivity and loading capacity are limited. In this work, we synthesized novel silica nanoparticles with uniform poly(2-(methacryloyloxy)ethyl)dimethyl-(3-sul-fopropyl)ammonium hydroxide (PMSA) brushes grafted onto the surface via reversible addition-fragmentation chain transfer (RAFT) polymerization (denoted as SiO2-RAFT@PMSA). The resulting SiO2-RAFT@PMSA nanoparticles demonstrated low detection limit (10fmol) and high recovery yield (over 88%) for glycopeptide enrichment from tryptic digest of human IgG. The SiO2-RAFT@PMSA nanoparticles were further applied for the analysis of mouse liver glycoproteome, a total number of 303 unique N-glycosylation sites corresponding to 185 glycoproteins was reliably profiled in three replicate nano-LC–MS/MS runs. Significantly, more glycopeptides were identified than those of nanoparticles, monolayer MSA molecules modified SiO2@single-MSA and nonuniform multi-layer PMSA brushes coated SiO2@PMSA, as well as commercial ZIC@HILIC beads and Click Maltose beads. The excellent performance of SiO2-RAFT@PMSA nanoparticles results from the non-fouling property, a large quantity of functional molecules and suitable link arms provided by uniform PMSA brushes, as well as efficient interaction between glycopeptides and uniform PMSA brushes. It is concluded that the synthesized SiO2-RAFT@PMSA nanoparticles exhibit great potential in glycoproteome analysis. Moreover, this strategy to modify nanopaticles with uniform polymer brushes via RAFT polymerization can also be explored to design other types of materials for bioseparation application.